Our experiments have shown that once the dynamics of the dyes and their flexible linkers are taken into account FRET efficiency measurements can produce quite accurate distance information. This was accomplished in three detailed studies, two using polyproline as a spacer of known length between donor and acceptor fluorophores, and one on the denatured state of a small single domain protein, the 66-residue alpha/beta protein L, for which the radius of gyration had been determined by small angle X-ray scattering. The interpretation of the experimental results for polyproline was aided enormously by atomistic molecular dynamics simulations of R. Best, that included the dyes and their long flexible linkers, and by nuclear magnetic resonance (NMR) measurements of A. Bax, which determined the fraction of cis isomers. This study finally settled the long-standing question of the origin of the width of FRET efficiency distributions in excess of that predicted by shot noise, a problem which has plagued experimentalists since the first single molecule experiments by Hochstrasser and Weiss and their coworkers. Comparison of the unfolded state sub-population of the 64 residue, alpha/beta protein L and the 66 residue, all-&#946; cold shock protein CspTm showed that the average radius of gyration (Rg) calculated from the FRET efficiency on freely diffusing molecules is identical for the two unfolded proteins at high denaturant concentration, consistent with the idea that under these conditions neither composition nor sequence matters. As the denaturant concentration is lowered the FRET efficiency increases signaling collapse. Analysis of the donor fluorescence decay of the unfolded subpopulation of both proteins indicates that chain dynamics is slow compared to the donor lifetime of 2 ns, later confirmed by others in direct measurements of this time.[unreadable] [unreadable] The 56-residue immunoglobulin binding domain of protein GB1 (protein G) was labeled with the FRET pairs and was attached to a surface in two ways, one via a 6-residue histidine tag to chelated copper ions embedded in a polyethylene glycol coated glass surface and another via a streptavidin-biotin conjugation. Photon trajectories of emitted donor and acceptor photons show simple behavior for the majority (80%) of the trajectories, i.e. the entire trajectory exhibits high FRET efficiency corresponding to the folded state as determined either by counting donor and acceptor photons or by obtaining the donor lifetime from the histogram of time delays between picosecond excitation and photon detection; the trajectory exhibits a low FRET efficiency trajectory corresponding to the unfolded state; or the trajectory exhibits both levels with one or more unresolvable jumps between them corresponding to folding or unfolding barrier-crossing events. The broad FRET efficiency distribution of the unfolded state arises mostly from the distribution of dye lifetimes and variable spectral shift from interaction of the dyes with the surface rather than from a distribution of unfolded conformations that inter-convert very slowly, as has been concluded for ribonuclease H in an earlier study. In spite of the dye-surface interactions, there is relatively little effect on the equilibrium and kinetic folding properties. Overall, our ability to obtain interpretable single molecule folding/unfolding trajectories represents a significant technological advance.